The Drosophila simulans clade diverged from the D. melanogaster lineage about 3 million years ago and radiated to produce D. simulans and D. sechellia. While D. melanogaster and D. simulans are generalists, D. sechellia only lays eggs on the morinda fruit. A key component of this ecological specialization involves evolution of the olfactory system. The odorant receptor field of the fruit fly is a model study of differentiation;62 different odorant receptor neurons are specified in close proximity to each other and are paired according to strict rules. In this proposal, I combine evolutionary genetics and developmental biology to approach the problem of differentiation. Olfactory neurons in fruit flies are found on the antenna and are housed in sensillae according to strict pairing rules. Each olfactory neuron expresses one unique odorant receptor (Or) that determines its response to different odors. I focus on two pairs of neurons;Or59b + Or85a-expressing neurons of the ab2 sensillum, and Or22a + Or85b-expressing neurons of the ab3 sensillum of D. melanogaster. Electrophysiology indicates that in D. sechellia, the number of ab3 sensillae has increased, and the ab2 sensillae are missing. I have extended these results by using GFP expression from D. melanogaster odorant receptor promoters in hybrids to either D. simulans or D. sechellia. I have found that expression of DmOr85a-GFP in neurons is lost, and I have confirmed the expansion of the ab3 neuron, Or22a in D. sechellia. However, I have found that these changes also occur in hybrids with D. simulans. My data indicate the presence of factors from D. simulans or D. sechellia that act in trans upon the DmOr22a and DmOr85a promoters. I will identify 1) elements in the D. melanogaster promoters that confer sensitivity to these trans-factors, and 2) the factors from D. simulans that affect Or22a and Or85a expression. To accomplish this I will determine the minimal promoter of each gene. I will then use a set of overlapping deletions to identify the promoter region that confers sensitivity to the trans- factors from D. simulans. Next, I will use this sequence to purify transcription factors in columns for protein sequencing. Finally, I will compare the expression of the factors that I identify between D. simulans and D. melanogaster. These experiments will reveal the identity of a gene that confers clade-specific olfactory patterning and suggest a mechanism for how a sensory system is able to adapt during evolution. This study will complement those that are restricted to the D. melanogaster model system, and will have implications for the general study of development and neuronal differentiation.